Summary of Study ST002422

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001559. The data can be accessed directly via it's Project DOI: 10.21228/M85X3W This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002422
Study Title	UBXD8 lipidomics from whole cells (Part 2)
Study Summary	The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipids found in mitochondria-associated membranes (MAM). LC-MS/MS lipidomics found significant changes in distinct lipid species in the MAM fraction of UBXD8 knockout cells. Our results suggest that lipids in MAM are regulated by UBXD8.
Institute	University of Arizona
Department	Immunobiology
Laboratory	Purdy Lab
Last Name	Purdy
First Name	John
Address	PO Box 245221, Tucson, Arizona, 85724, USA
Email	purdylab@gmail.com
Phone	520-626-4371
Submit Date	2023-01-01
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-01-16
Release Version	1

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Project:

Project ID:	PR001559
Project DOI:	doi: 10.21228/M85X3W
Project Title:	UBXD8 lipidomics from whole cells
Project Summary:	The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct lipid species in UBXD8 knockout cells, in particular in saturated or mono-unsaturated lipid species. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark in a variety of human disorders such as neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on UBXD8.
Institute:	University of Arizona
Department:	Immunobiology
Laboratory:	Purdy Lab
Last Name:	Purdy
First Name:	John
Address:	PO Box 245221, Tucson, Arizona, 85724, USA
Email:	purdylab@gmail.com
Phone:	520-626-4371
Funding Source:	NIH R01 AI162671
Contributors:	Rakesh Ganji, Joao A. Paulo, Yuecheng Xi, Ian Kline, Jiang Zhu, Christoph S. Clemen, Conrad C. Weihl, John G. Purdy, Steve P. Gygi, and Malavika Raman

Subject:

Subject ID:	SU002511
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	HEK293T

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Description	inj vol
SA242379	20220820_tufts_MAMfrac_neg_rep8_KO_10ul	UBXD8 Knockout	10
SA242380	20220508_neg_KO_rep4_10ul	UBXD8 Knockout	10
SA242381	20220508_neg_KO_rep3_10ul	UBXD8 Knockout	10
SA242382	20220508_neg_KO_rep1_10ul	UBXD8 Knockout	10
SA242383	20220820_tufts_MAMfrac_neg_rep8_KO_15ul	UBXD8 Knockout	15
SA242384	20220508_pos_KO_rep3_16ul	UBXD8 Knockout	16
SA242385	20220820_tufts_MAMfrac_pos_rep8_KO_16ul	UBXD8 Knockout	16
SA242386	20220508_pos_KO_rep4_16ul	UBXD8 Knockout	16
SA242387	20220508_pos_KO_rep1_16ul	UBXD8 Knockout	16
SA242388	20220820_tufts_MAMfrac_pos_rep8_KO_4ul	UBXD8 Knockout	4
SA242389	20220508_pos_KO_rep3_4ul	UBXD8 Knockout	4
SA242390	20220508_pos_KO_rep1_4ul	UBXD8 Knockout	4
SA242391	20220508_pos_KO_rep4_4ul	UBXD8 Knockout	4
SA242392	20220820_tufts_MAMfrac_neg_rep8_KO_5ul	UBXD8 Knockout	5
SA242393	20220508_pos_KO_rep1_8ul	UBXD8 Knockout	8
SA242394	20220508_pos_KO_rep4_8ul	UBXD8 Knockout	8
SA242395	20220820_tufts_MAMfrac_pos_rep8_KO_8ul	UBXD8 Knockout	8
SA242396	20220508_pos_KO_rep3_8ul	UBXD8 Knockout	8
SA242397	20220508_neg_WT_rep3_10ul	Wild-type	10
SA242398	20220508_neg_WT_rep4_10ul	Wild-type	10
SA242399	20220508_neg_WT_rep1_10ul	Wild-type	10
SA242400	20220820_tufts_MAMfrac_neg_rep8_WT_10ul	Wild-type	10
SA242401	20220820_tufts_MAMfrac_neg_rep8_WT_15ul	Wild-type	15
SA242402	20220508_pos_WT_rep3_16ul	Wild-type	16
SA242403	20220508_pos_WT_rep1_16ul	Wild-type	16
SA242404	20220508_pos_WT_rep4_16ul	Wild-type	16
SA242405	20220820_tufts_MAMfrac_pos_rep8_WT_16ul	Wild-type	16
SA242406	20220820_tufts_MAMfrac_pos_rep8_WT_4ul	Wild-type	4
SA242407	20220508_pos_WT_rep3_4ul	Wild-type	4
SA242408	20220508_pos_WT_rep4_4ul	Wild-type	4
SA242409	20220508_pos_WT_rep1_4ul	Wild-type	4
SA242410	20220820_tufts_MAMfrac_neg_rep8_WT_5ul	Wild-type	5
SA242411	20220508_pos_WT_rep1_8ul	Wild-type	8
SA242412	20220508_pos_WT_rep3_8ul	Wild-type	8
SA242413	20220508_pos_WT_rep4_8ul	Wild-type	8
SA242414	20220820_tufts_MAMfrac_pos_rep8_WT_8ul	Wild-type	8

Showing results 1 to 36 of 36

Showing results 1 to 36 of 36

Collection:

Collection ID:	CO002504
Collection Summary:	Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and streptomycin. Cells were maintained in a humidified, 5 % CO2 atmosphere at 37°C. The CRISPR-Cas9 gene editing system was used to generate UBXD8 knockout cell lines in HEK293T cells. Cells were seeded into four 150 mm TC dishes. Cells were lysed in Homogenization buffer (225 mM mannitol, 75 mM sucrose, and 30 mM Tris-Cl, pH 7.4) using a Dounce homogenizer. The lysate was centrifuged three times at 600xg for 5 minutes to remove unlysed cells and nuclei resulting in post-nuclear supernatants (PNS). The cleared lysate was centrifuged at 7000xg to separate crude mitochondrial pellet and supernatant containing microsomes. The supernatant was cleared by centrifugation at 20,000xg for 30 minutes followed by microsome isolation using high-speed centrifugation at 100,000xg for 1 hour. The crude mitochondrial pellet was washed twice in homogenization buffer containing 0.1 mM EGTA at 7000xg and 10,000xg for 10 minutes. MAMs were isolated from crude mitochondria using 30 % Percoll gradient centrifugation at 95,000xg for 1 hr in a swinging-bucket rotor. The banded MAM fraction was washed once with phosphate-buffered saline (PBS) before lysing in lysis buffer (50 mM Tris-Cl, pH 7.2, 150 mM NaCl). The pure mitochondrial fractions were resuspended and washed in mitochondrial resuspension buffer (250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES pH7.4). Protein concentrations for both soluble and pellet fractions were determined by DC protein assay kit (Biorad). For lipidomics of MAMs, the final banded MAM fraction was washed twice with liquid chromatography-mass spectrometry (LC-MS) grade PBS and the final MAM pellet was resuspended in LC-MS grade PBS. Next, 1mL of cold 50% methanol was added and the MAMS were transferred to glass vials. Chloroform was added (0.5mL) and the mixture was gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration. Extracted lipids were resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis.
Sample Type:	Epithelial cells

Treatment:

Treatment ID:	TR002523
Treatment Summary:	Wild-type vs UBXD8 Knockout

Sample Preparation:

Sampleprep ID:	SP002517
Sampleprep Summary:	Lipids were isolated from collected cultured cells. Cells were washed with PBS, treated with cold 50% methanol (1mL) and transferred to glass vials. Next, chloroform (0.5mL) was added and samples were gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration when resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. To assist quantification various volumes were injected for MS (4, 8, and 16ul for positive mode analysis and 5, 10, and 15ul for negative mode analysis).

Sampleprep ID:

SP002517

Sampleprep Summary:

Lipids were isolated from collected cultured cells. Cells were washed with PBS, treated with cold 50% methanol (1mL) and transferred to glass vials. Next, chloroform (0.5mL) was added and samples were gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration when resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. To assist quantification various volumes were injected for MS (4, 8, and 16ul for positive mode analysis and 5, 10, and 15ul for negative mode analysis).

Combined analysis:

Analysis ID	AN003944	AN003945
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um)	Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH002920
Chromatography Summary:	Lipids were identified and quantitatively measured using ultra high-performance liquid-chromatography high-resolution tandem MS/MS (UHPLC-MS/MS) as recently described89,90. Separation of lipids was done by reverse-phase chromatography using a Waters ACQUITY CSH C18 column (150x2.1mm; 1.7um) at 60°C using a Vanquish UHPLC system (Thermo Scientific) and two solvents: solvent A (40:60 water-methanol plus 10mM ammonium formate and 0.1% formic acid) and solvent B (10:90 methanol-isopropanol plus 10mM ammonium formate and 0.1% formic acid). UHPLC was performed at a 0.25 ml per min flow rate for 30 min per sample, starting at 25% solvent B and ending at 100% solvent B as described. The column was washed and equilibrated between samples. Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Each sample was repeatedly analyzed using various injection volumes (4, 8, 16ul for positive mode and 5, 10, and 15ul for negative mode).
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um)
Column Temperature:	60
Flow Gradient:	25% to 100%
Flow Rate:	0.25mL per min
Solvent A:	40% water; 60% methanol 10mM ammonium formate and 0.1% formic acid
Solvent B:	10% methanol; 90% isopropanol 10mM ammonium formate and 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003680
Analysis ID:	AN003944
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and El-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive).
Ion Mode:	POSITIVE

MS ID:	MS003681
Analysis ID:	AN003945
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and El-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive).
Ion Mode:	NEGATIVE